Self-Inactivating lentiviral vectors resist proviral methylation but do not confer position-independent expression in hematopoietic stem cells

Azim Mohamedali, Francois Moreau-Gaudry, Emmanuel Richard, Ping Xia, Jan Nolta, Punam Malik

Research output: Contribution to journalArticle

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Abstract

Oncoretroviral expression is transcriptionally silenced in embryonic and hematopoietic stem cells (HSCs). This is associated with methylation of viral and internal promoters. We determined whether self-inactivating (SIN) lentiviral vectors (LV) would circumvent proviral silencing in HSCs. We studied long-term expression, methylation, and position effects (PE) from two GFP-encoding SIN-LV containing erythroid enhancers and the human ankyrin-1 promoter (h-Ank-P) using the murine secondary bone marrow (BM) transplant assay. Proviral expression was detected in RBC 6-11 months following transplant only in 28 of 49 secondary mice, with 0.9 ± 0.2 copy/cell and oligoclonally integrated provirus in BM, spleen, and thymus. Twenty-one of 49 secondary mice lacked integrated provirus. Secondary mice containing provirus also had GFP-expressing RBCs, although proviral copy number did not always correlate with expression, suggesting either proviral methylation or chromatin PE. The endogenous h-Ank-P was partially methylated in nonerythroid cell lines and unmethylated in erythroid cell lines. However, h-Ank-P in the provirus was unmethylated in erythroid and nonerythroid cells within secondary murine BM. Despite lack of methylation, GFP expression was variable in secondary BFU-e and in single-copy mouse erythroleukemia cell clones. Taken together, these data show that erythroid-specific SIN-LV express long term and resist methylation-associated proviral silencing, but may require additional elements to confer position-independent expression.

Original languageEnglish (US)
Pages (from-to)249-259
Number of pages11
JournalMolecular Therapy
Volume10
Issue number2
DOIs
StatePublished - Aug 2004
Externally publishedYes

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Hematopoietic Stem Cells
Proviruses
Methylation
Ankyrins
Erythroid Cells
Bone Marrow
Transplants
Cell Line
Leukemia, Erythroblastic, Acute
Embryonic Stem Cells
Thymus Gland
Chromatin
Spleen
Clone Cells

ASJC Scopus subject areas

  • Molecular Biology

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Self-Inactivating lentiviral vectors resist proviral methylation but do not confer position-independent expression in hematopoietic stem cells. / Mohamedali, Azim; Moreau-Gaudry, Francois; Richard, Emmanuel; Xia, Ping; Nolta, Jan; Malik, Punam.

In: Molecular Therapy, Vol. 10, No. 2, 08.2004, p. 249-259.

Research output: Contribution to journalArticle

Mohamedali, Azim ; Moreau-Gaudry, Francois ; Richard, Emmanuel ; Xia, Ping ; Nolta, Jan ; Malik, Punam. / Self-Inactivating lentiviral vectors resist proviral methylation but do not confer position-independent expression in hematopoietic stem cells. In: Molecular Therapy. 2004 ; Vol. 10, No. 2. pp. 249-259.
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